During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge.
Copper-free click chemistry is a fairly new chemistry that has been commercialized during the past few years. More and more click chemistry-based reagents are becoming available commercially which makes the formulation development much easier for scientists. The great advantage of this chemistry is biocompatibility since no cytotoxic copper catalyst is required. By far, click chemistry is the most efficient and easiest conjugation chemistry available for coupling of antibodies and other reactive ligands to the surface of the liposomes. The conjugation chemistry is based on the reaction of the dibenzocyclooctyne (DBCO) reagent with an azide linker to form a stable triazole. DBCO moiety can be on the antibody and azide moiety can be on liposomes and vice versa. This conjugation protocol is based on the reaction of the dibenzocyclooctyne (DBCO) group of the liposomes with an azide linker on the antibody, peptide or proteins.
There are many commercialized reagents that can be used for azide modification of proteins, peptides and antibodies. To see the list of commercialized reagents for azide modification see here.
For other reactive (PEGylated and non-PEGyalated products) ImmunoFluor™ products suitable for other types conjugation methods see here.
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加尾法是采用加A酶先对mirna进行加尾，然后再用带oligodt的引物反转录，他的反转录引物是通用的，一次反转录可以获得所有miRNA的cdna，效率高。茎环法是采用特异性反转录引物序列+颈环结构作为反转录引物进行反转录的，一次反转录只能获得一种mirna的cdna，可能有几种一起反转录的，这个我不太清楚一起反转录的效果。然后茎环法的经典即ABI的探针法定量试剂盒。我用过茎环法反转录+染料法定量的，下游引物为通用引物，结果不太好，就现在用的这家的他采用的是双向引物都是特异性的，效果还可以。不知道我说的清楚不，希望能对你有帮助。
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBRGreenMix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBRGreen，并且如果你那是ABI或者Stratagene的PCR如果用SYBRGreen还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
